1. Technical Field
The present invention pertains generally to viral proteins. In particular, the invention relates to truncated, secreted forms of hepatitis C virus E1 and E2 proteins and the isolation and recombinant production of the same.
2. Background of the Invention
Hepatitis C Virus (HCV) is the principal cause of parenteral non-A, non-B hepatitis which is transmitted largely through blood transfusion and sexual contact. The virus is present in between 0.4 to 2.0% of blood donors. Chronic hepatitis develops in approximately 50% of infections and of these, approximately 20% of infected individuals develop liver cirrhosis which sometimes leads to hepatocellular carcinoma. Accordingly, the study and control of the disease is of medical importance.
The viral genomic sequence of HCV is known, as are methods for obtaining the sequence. See, e.g., International Publication Nos. WO 89/04669; WO 90/11089; and WO 90/14436. In particular, HCV has a 9.5 kb positive-sense, single-stranded RNA genome and is a member of the Flaviridae family of viruses. Currently, there are 6 distinct, but related genotypes of HCV based on phylogenetic analyses (Simmonds et al., J. Gen. Virol. (1993) 74:2391-2399). The virus encodes a single polyprotein having more than 3000 amino acid residues (Choo et al., Science (1989) 244:359-362; Choo et al., Proc. Natl. Acad. Sci. USA (1991) 88:2451-2455; Han et al., Proc. Natl. Acad. Sci. USA (1991) 88:1711-1715). The polyprotein is processed co- and post-translationally into both structural and non-structural (NS) proteins.
In particular, there are three putative structural proteins, consisting of the N-terminal nucleocapsid protein (termed "core") and two envelope glycoproteins, "E1" (also known as E) and "E2" (also known as E2/NS1). (See, Houghton et al., Hepatology (1991) 14:381-388, for a discussion of HCV proteins, including E1 and E2.) E1 is detected as a 32-35 kDa species and is converted into a single endo H-sensitive band of approximately 18 kDa. By contrast, E2 displays a complex pattern upon immunoprecipitation consistent with the generation of multiple species (Grakoui et al., J. Virol. (1993) 67:1385-1395; Tomei et al., J. Virol. (1993) 67:4017-4026.). The HCV envelope glycoproteins E1 and E2 form a stable complex that is co-immunoprecipitable (Grakoui et al., J. Virol. (1993) 67:1385-1395; Lanford et al., Virology (1993) 197:225-235; Ralston et al., J. Virol. (1993) 67:6753-6761). The HCV E1 and E2 glycoproteins are of considerable interest because they have been shown to be protective in primate studies. (Choo et al., Proc. Natl. Acad. Sci. USA (1994) 91:1294-1298).
The envelope of the HCV virion remains uncharacterized. Thus, expression studies using recombinant cDNA templates are the only means currently available to study envelope biosynthesis. E1 and E2 are retained within cells and lack complex carbohydrate when expressed stably or in a transient Vaccinia virus system (Spaete et al., Virology (1992) 188:819-830; Ralston et al., J. Virol. (1993) 67:6753-6761). Since the E1 and E2 proteins are normally membrane-bound in these expression systems, it would be desirable to produce secreted forms to facilitate purification of the proteins for further use.
It has been found that removal of the transmembrane domain of the viral cell surface glycoproteins of influenza virus (Sveda, et al., Cell (1982) 30:649-656; Gething and Sambrook, Nature (1982) 300:598-603) and vesicular stomatitis virus (Rose and Bergmann, Cell (1982) 30:753-762) results in secretion of the truncated glycoprotein from mammalian host cells. See also EPO Publication No. 139,417. Similarly, truncated cytomegalovirus gH is secreted when expressed in baculovirus cells (International Publication No. WO 92/02628, published Feb. 20, 1992). A C-terminally truncated HCV E2 molecule, capable of secretion from mammalian cells, has been described. Spaete et al., Virology (1992) 188:819-830 However, the transmembrane anchor region of E1 has not heretofore been elucidated and hence the production of truncated forms of HCV E1 for secretion has not been previously disclosed. Furthermore, complexes of truncated, secreted E1 and E2 polypeptides have not been previously described.